Anti-Caspase-3 antibody [EPR18297]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Immunohistochemical analysis of paraffin-embedded Human cervical cancer tissue labeling active and pro Caspase 3 with ab184787 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus and cytoplasm staining on tumor cells of Human cervix cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling active and pro Caspase 3 with ab184787 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nucleus and cytoplasm staining on lymphocytes of tonsil is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling Caspase-3 with ab184787 at a concentration of 0.05µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab184787 Anti-Caspase-3 antibody [EPR18297] antibody was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• IP

Supplier Data

Immunoprecipitation - Anti-Caspase-3 antibody [EPR18297] (AB184787)

active and pro Caspase 3 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate treated with 1uM staurosporine for 4 hours with ab184787 at 1/80 dilution. Western blot was performed from the immunoprecipitate using ab184787 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/1500 dilution.

Lane 1 : HeLa whole cell lysate treated with 1uM staurosporine for 4 hours 10 μg (Input).

Lane 2 : ab184787 IP in HeLa whole cell lysate treated with 1uM staurosporine for 4 hours.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab184787 in HeLa whole cell lysate treated with 1uM staurosporine for 4 hours.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Caspase-3 antibody [EPR18297] (ab184787)

Predicted band size: 31 kDa

Observed band size: 17 kDa,32 kDa

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• WB

Supplier Data

Western blot - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Blocking/Dilution buffer : 5% NFDM/TBST.

Specificity : interacts with full length pro-Caspase 3 and the p17 subunit.

The Caspase-3 precursor is first cleaved between D175 and S176 to produce the p11 subunit and p20 fragment. Subsequently, the p20 fragment is cleaved between D28 and S29 to generate the p17 subunit (Proc. Natl. Acad. Sci. USA. 93, 7464-7469 - PMID : 8755496).

All lanes:

Western blot - Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/2000 dilution

Lane 1:

Untreated Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 10 µg

Lane 2:

Jurkat whole cell lysates treated with 1uM staurosporine for 4 hours at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 31 kDa

Observed band size: 17 kDa,32 kDa

false

Exposure time: 1min

• WB

Lab

Western blot - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Lane 1 : Wild-type HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 2 : Wild-type HAP1 cell lysate
Lane 3 : Caspase-3 knockout HAP1 cell lysate + Staurosporine (1μM for 4h)
Lane 4 : Caspase-3 knockout HAP1 cell lysate
Lanes 1 - 4 : Merged signal (red and green). Green - ab184787 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab184787 was shown to recognise pro Caspase 3 when Caspase 3 knockout samples were used, along with additional cross-reactive bands. Wild-type and Caspase 3 knockout samples (± Staurosporine treatment) were subjected to SDS-PAGE. ab184787 and ab8245 (loading control to GAPDH) were diluted to 1/2000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776)
secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Caspase-3 antibody [EPR18297] (ab184787)

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Anti-CASP3 antibody [EPR18297] (ab184787) staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab184787 was shown to bind specifically to CASP3. A band was observed at 35 kDa in treated wild-type HAP1 and HeLa cell lysates with no signal observed at this size in CASP3 knockout HAP1 cell line and a band at a lower molecular weight in the CAPS3 knockout HeLa cell line ab255370 (knockout cell lysate ab263779) which cannot be cleaved to active CASP3. To generate this image, wild-type and CASP3 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/2000 dilution

Lane 1:

Wild-type HAP1 Treated Staurosporine (2 uM, 4h) cell lysate at 20 µg

Lane 2:

CASP3 knockout HAP1 Treated Staurosporine (2 uM, 4h) cell lysate at 20 µg

Lane 3:

Wild-type HAP1 Vehicle Control Staurosporine (0 uM, 4h) cell lysate at 20 µg

Lane 4:

CASP3 knockout HAP1 Vehicle Control Staurosporine (0 uM, 4h) cell lysate at 20 µg

Lane 5:

Wild-type HeLa Treated Staurosporine (2 uM, 4h) cell lysate at 20 µg

Lane 6:

CASP3 knockout HeLa Treated Staurosporine (2 uM, 4h) cell lysate at 20 µg

Lane 7:

Wild-type HeLa Vehicle Control Staurosporine (0 uM, 4h) cell lysate at 20 µg

Lane 8:

CASP3 knockout HeLa Vehicle Control Staurosporine (0 uM, 4h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 31 kDa

Observed band size: 35 kDa

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• WB

Lab

Western blot - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control. Bands between 27-32kDa represent cleavage of the procaspase at D9 and D28, respectively (PMID : 14567691)

All lanes:

Western blot - Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/1000 dilution

Lane 1:

Mouse Alzheimer's disease brain tissue lysate at 20 µg

Lane 2:

Mouse brain cancer tissue lysate at 20 µg

Lane 3:

Mouse hippocampus tissue lysate at 20 µg

Lane 4:

Mouse spinal cord tissue lysate at 20 µg

Lane 5:

Mouse cerebellum tissue lysate at 20 µg

Lane 6:

Mouse cerebral cortex tissue lysate at 20 µg

Lane 7:

Mouse hypothalamus tissue lysate at 20 µg

Lane 8:

Mouse heart tissue lysate at 20 µg

Lane 9:

Mouse liver tissue lysate at 20 µg

Lane 10:

Human brain tissue lysate at 20 µg

Lane 11:

Human liver tissue lysate at 20 µg

Lane 12:

Human hypothalamus tissue lysate at 20 µg

Lane 13:

Human heart tissue lysate at 20 µg

Lane 14:

Human cerebellum tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 32 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Blocking and diluting buffer : 5% NFDM/TBST.

ab184787 recognizes pro-Caspase 3 and unable to detect the active caspases after induction in mouse and rat samples.

All lanes:

Western blot - Anti-Caspase-3 antibody [EPR18297] (ab184787) at 0.7 µg/mL

Lane 1:

Untreated NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) treated with 1μM Staurosporine for 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-Caspase-3 antibody [EPR18297] (AB184787)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as GAPDH loading control. Bands between 27-32kDa represent cleavage of the procaspase at D9 and D28, respectively (PMID : 14567691)

All lanes:

Western blot - Anti-Caspase-3 antibody [EPR18297] (ab184787) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat hippocampus tissue lysate at 20 µg

Lane 3:

Rat spinal cord tissue lysate at 20 µg

Lane 4:

Rat cerebellum tissue lysate at 20 µg

Lane 5:

Rat cerebral cortex tissue lysate at 20 µg

Lane 6:

Rat hypothalamus tissue lysate at 20 µg

Lane 7:

Rat heart tissue lysate at 20 µg

Lane 8:

Rat liver tissue lysate at 20 µg

Lane 9:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 31 kDa

false

Exposure time: 20s